Method of isolating cytotoxic heterocomplex associated with multiple sclerosis

Abstract

The invention relates to an isolated cytotoxic factor which is associated with multiple sclerosis and which is selected from the heterocomplex GM2AP/GM2/MRP14 and mutated GM2AP/GM2/MRP14, and to the method of detecting said factor in a biological sample to be tested. The inventive method comprises the following steps consisting in: (i) bringing the biological sample into contact with at least one capture antibody selected from antibodies that bind specifically to the GM2AP protein, to the mutated GM2AP protein, to the MRP14 protein, to the complex GM2AP/GM2, to the complex mutated GM2AP/GM2 and to the complex MRP14/GM2, and with at least one labeled detection antibody selected from antibodies that bind specifically to the GM2AP protein, to the mutated GM2AP protein, to the MRP14 protein, to the complex GM2AP/GM2, to the complex mutated GM2AP/GM2 and to the complex MRP14/GM2, and (ii) detecting and/or quantifying the cytotoxic factor by detecting and/or quantifying the labeled detection antibody.

1. A method specifically for detecting and/or quantifying a cytotoxic factor having a gliotoxic activity, associated with multiple sclerosis, in a biological sample, comprising:
 isolating a heterocomplex from the biological sample, wherein the heterocomplex is chosen from a GM2AP/GM2/MRP14 heterocomplex and a mutated GM2AP/GM2/MRP14 heterocomplex in which mutated GM2AP has the amino acid sequence set forth in SEQ ID NO:2; 
 wherein:
 the heterocomplex is isolated by means of at least one antibody that binds specifically to the heterocomplex, and 
 the cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the antibody. 
 
 
     
     
       2. The method as claimed in  claim 1 , in which the biological sample is selected from the group consisting of serum, plasma, urine and cerebrospinal fluid. 
     
     
       3. The method as claimed in  claim 1 , according to which the heterocomplex is isolated by means of at least two antibodies that bind specifically to the heterocomplex, and said cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the two antibodies. 
     
     
       4. The method as claimed in  claim 3 , according to which the heterocomplex is isolated by means of at least two antibodies, at least one of which binds specifically to GM2AP or mutated GM2AP of the heterocomplex, and at least the other of which binds specifically to MRP14 of the heterocomplex, and said cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the two antibodies. 
     
     
       5. The method as claimed in  claim 4 , according to which at least one of said antibodies is a capture antibody and at least one of said antibodies is a detection antibody. 
     
     
       6. The method of  claim 1 , wherein the biological sample is subjected to the following treatments prior to the step of isolating:
 digesting the proteins of the sample with proteinase K, 
 inactivating the proteinase K, and 
 neutralizing the pH. 
 
     
     
       7. The method as claimed in  claim 6 , wherein inactivating the proteinase K is carried out by precipitation with trichloroacetic acid, and wherein neutralizing the pH is carried out by the addition of a tris-maleate buffer. 
     
     
       8. A method for detecting and/or quantifying a cytotoxic factor having a gliotoxic activity and associated with multiple sclerosis, comprising:
 providing a biological sample, 
 digesting the proteins of the sample with proteinase K, 
 inactivating the proteinase K, 
 neutralizing the pH, and 
 isolating a heterocomplex from the biological sample, wherein the heterocomplex is chosen from a GM2AP/GM2/MRP14 heterocomplex and a mutated GM2AP/GM2/MRP14 heterocomplex in which mutated GM2AP has the amino acid sequence set forth in SEQ ID NO:2. 
 
     
     
       9. The method of  claim 8 , wherein:
 inactivating the proteinase K is carried out by precipitation with trichioroacetic acid, and 
 neutralizing the pH is carried out by the addition of a tris-maleate buffer. 
 
     
     
       10. The method as claimed in  claim 8 , wherein the biological sample is selected from the group consisting of serum, plasma, urine, and cerebrospinal fluid. 
     
     
       11. The method of  claim 8 , wherein:
 the heterocomplex is isolated by means of at least one antibody that binds specifically to the heterocomplex, and 
 the cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the antibody. 
 
     
     
       12. The method of  claim 8 , wherein:
 the heterocomplex is isolated by means of at least two antibodies that bind specifically to the heterocomplex, and 
 the cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the two antibodies. 
 
     
     
       13. The method of  claim 12 , wherein at least one of the antibodies is a capture antibody and at least one antibody is a detection antibody. 
     
     
       14. The method of  claim 8 , wherein:
 the heterocomplex is isolated by means of at least two antibodies, at least one of which binds specifically to GM2AP or mutated GM2AP of the heterocomplex, and at least the other of which binds specifically to MRP14 of the heterocomplex, and 
 the cytotoxic factor is detected and/or quantified by demonstrating the formation of a complex consisting of the heterocomplex and the two antibodies. 
 
     
     
       15. A composition for detecting and/or quantifying a cytotoxic factor associated with multiple sclerosis comprising at least one antibody that specifically binds to a heterocomplex selected from the group consisting of:
 a GM2AP/GM2/MRP14 heterocomplex and 
 a mutated GM2AP/GM2/MRP14 heterocomplex in which mutated GM2AP has the amino acid sequence set forth in SEQ ID NO:2. 
 
     
     
       16. The composition as claimed in  claim 15 , comprising at least two antibodies that bind specifically to the heterocomplex.